State-of-the-Art Cancer Biology, Biodiagnostics and Therapeutics in Japan.

Early cancer detection is key to improving patient survival and quality of life and reducing cancer treatments' financial burden [...].

Pancreatic Cancer and Detection Methods.

The pancreas is a vital organ with exocrine and endocrine functions. Pancreatitis is an inflammation of the pancreas caused by alcohol consumption and gallstones. This condition can heighten the risk of pancreatic cancer (PC), a challenging disease with a high mortality rate. Genetic and epigenetic factors contribute significantly to PC development, along with other risk factors. Early detection is crucial for improving PC outcomes. Diagnostic methods, including imagining modalities and tissue biopsy, aid in the detection and analysis of PC. In contrast, liquid biopsy (LB) shows promise in early tumor detection by assessing biomarkers in bodily fluids. Understanding the function of the pancreas, associated diseases, risk factors, and available diagnostic methods is essential for effective management and early PC detection. The current clinical examination of PC is challenging due to its asymptomatic early stages and limitations of highly precise diagnostics. Screening is recommended for high-risk populations and individuals with potential benign tumors. Among various PC screening methods, the N-NOSE plus pancreas test stands out with its high AUC of 0.865. Compared to other commercial products, the N-NOSE plus pancreas test offers a cost-effective solution for early detection. However, additional diagnostic tests are required for confirmation. Further research, validation, and the development of non-invasive screening methods and standardized scoring systems are crucial to enhance PC detection and improve patient outcomes. This review outlines the context of pancreatic cancer and the challenges for early detection.

Clinical Possibility of Caenorhabditis elegans as a Novel Evaluation Tool for Esophageal Cancer Patients Receiving Chemotherapy: A Prospective Study.

BACKGROUND: The nematode Caenorhabditis elegans (C. elegans) possesses a sophisticated sense of smell and is used for a novel cancer screening test that utilizes the chemotaxis index. We designed a single-institution, prospective study to confirm the ability of Nematode Nose (N-NOSE) to determine preoperative chemotherapy's efficacy for esophageal cancer patients. PATIENTS AND METHODS: We investigated the predictability of N-NOSE screening for the clinical effects of preoperative chemotherapy for esophageal cancer patients receiving radical surgery. The index reduction score (IRS) was calculated via the chemotaxis of C. elegans at three points: before treatment, before surgery, and after surgery, and its clinical relevance was examined. RESULT: Thirty-nine patients with esophageal cancer were enrolled from August 2020 to December 2021, and 30 patients receiving radical surgery were examined. Complete response or partial response was achieved in 23 cases (76.7%). When the target of the treatment effect was complete response only, the prediction accuracies of the IRS calculated by area under the curve was 0.85 (95% Confidence interval: 0.62-1) in clinically achieving complete response group, and the sensitivity and specificity were 1 and 0.63, respectively. CONCLUSION: Index reduction score using N-NOSE screening may reflect the efficacy of chemotherapy for esophageal cancer patients. A large-scale prospective study at multiple centers is desired in the future.

C. elegans as a Powerful Tool for Cancer Screening.

Regular cancer screening is critical for early cancer detection. Cancer screening tends to be burdensome, invasive, and expensive, especially for a comprehensive multi-organ check. Improving the rate and effectiveness of routine cancer screenings remain a challenge in health care. Multi-cancer early detection (MCED) is an exciting concept and a potentially effective solution for addressing current issues with routine cancer screening. In recent years, several technologies have matured for MCED, such as identifying cell-free tumor DNA in blood or using organisms such as Caenorhabditis elegans as a tool for early cancer detection. In Japan, N-NOSE is a commercially available multi-cancer detection test based on the chemotaxis of C. elegans using a urine sample showing 87.5% sensitivity and 90.2% specificity. In this review, we focus on using C. elegans as a powerful biosensor for universal cancer screening. We review N-NOSE clinical research results, spotlighting it as an effective primary cancer screening test.

Pancreatic Cancer Research beyond DNA Mutations.

Pancreatic ductal adenocarcinoma (PDAC) is caused by genetic mutations in four genes: KRAS proto-oncogene and GTPase (KRAS), tumor protein P53 (TP53), cyclin-dependent kinase inhibitor 2A (CDKN2A), and mothers against decapentaplegic homolog 4 (SMAD4), also called the big 4. The changes in tumors are very complex, making their characterization in the early stages challenging. Therefore, the development of innovative therapeutic approaches is desirable. The key to overcoming PDAC is diagnosing it in the early stages. Therefore, recent studies have investigated the multifaced characteristics of PDAC, which includes cancer cell metabolism, mesenchymal cells including cancer-associated fibroblasts and immune cells, and metagenomics, which extend to characterize various biomolecules including RNAs and volatile organic compounds. Various alterations in the KRAS-dependent as well as KRAS-independent pathways are involved in the refractoriness of PDAC. The optimal combination of these new technologies is expected to help treat intractable pancreatic cancer.

A new detection method for canine and feline cancer using the olfactory system of nematodes.

Cancer is the leading cause of death in dogs and cats. Early diagnosis of cancer is critical for effective treatment and improving survival rates. Nematode-NOSE (N-NOSE) is a commercially available non-invasive human cancer screening test that uses the sense of smell of the nematode Caenorhabditis elegans showing a distinct chemotactic response toward the urine of an individual with cancer compared to healthy ones. 15 types of human cancer (stomach, colon-rectum, lung, breast, pancreas, liver, prostate, uterus, esophagus, gallbladder, bile duct, kidney, urinary bladder, ovary, and oropharynx cancers) can be detected by N-NOSE. A non-invasive method for accurate cancer screening is needed for pets. In this study, we evaluated the effectiveness of N-NOSE in detecting cancer using canine and feline urine samples. We found a significant difference in chemotaxis index values between healthy subjects and cancer patients in both canine (p < 0.01*) and feline (p < 0.04*) urine samples. Receiver operating characteristic (ROC) analysis highlights the good performance of the test with areas under the curve (AUC) of 0.8114 and 0.7851 for dogs and 0.7667 and 0.9000 for cats when using 2 different dilutions of urine samples. Our study suggests that N-NOSE has the potential as a simple, accurate, and low-cost cancer screening test in both dogs and cats.

Nematode-Applied Technology for Human Tumor Microenvironment Research and Development.

Nematodes, such as Caenorhabditis elegans, have been instrumental to the study of cancer. Recently, their significance as powerful cancer biodiagnostic tools has emerged, but also for mechanism analysis and drug discovery. It is expected that nematode-applied technology will facilitate research and development on the human tumor microenvironment. In the history of cancer research, which has been spurred by numerous discoveries since the last century, nematodes have been important model organisms for the discovery of cancer microenvironment. First, microRNAs (miRNAs), which are noncoding small RNAs that exert various functions to control cell differentiation, were first discovered in C. elegans and have been actively incorporated into cancer research, especially in the study of cancer genome defects. Second, the excellent sense of smell of nematodes has been applied to the diagnosis of diseases, especially refractory tumors, such as human pancreatic cancer, by sensing complex volatile compounds derived from heterogeneous cancer microenvironment, which are difficult to analyze using ordinary analytical methods. Third, a nematode model system can help evaluate invadosomes, the phenomenon of cell invasion by direct observation, which has provided a new direction for cancer research by contributing to the elucidation of complex cell-cell communications. In this cutting-edge review, we highlight milestones in cancer research history and, from a unique viewpoint, focus on recent information on the contributions of nematodes in cancer research towards precision medicine in humans.

Scent test using Caenorhabditis elegans to screen for early-stage pancreatic cancer.

Although early detection and diagnosis are indispensable for improving the prognosis of patients with pancreatic cancer, both have yet to be achieved. Except for pancreatic cancer, other cancers have already been screened through scent tests using animals or microorganisms, including Caenorhabditis elegans. While such a method may greatly improve the prognosis of pancreatic cancer, no studies have investigated the same, mainly given the difficulty of collecting suitable samples from patients with early-stage pancreatic cancer. In this study, we organized a nationwide study group comprising high-volume centers throughout Japan to collect patients with very-early-stage pancreatic cancer (stage 0 or IA). We initially performed an open-label study involving 83 cases (stage 0-IV), with subsequent results showing significant differences after surgical removal in stage 0-IA (×10 dilution: p < 0.001; ×100 dilution: p < 0.001). Thereafter, a blinded study on 28 cases (11 patients with stage 0 or IA disease and 17 healthy volunteers) was conducted by comparing very-early-stage pancreatic cancer patients with healthy volunteers to determine whether C. elegans could detect the scent of cancer for the diagnosis of early-stage pancreatic cancer. Preoperative urine samples had a significantly higher chemotaxis index compared to postoperative samples in patients with pancreatic cancer [×10 dilution: p < 0.001, area under the receiver operating characteristic curve (AUC) = 0.845; ×100 dilution: p < 0.001, AUC = 0.820] and healthy volunteers (×10 dilution: p = 0.034; ×100 dilution: p = 0.088). Moreover, using the changes in preoperative and postoperative chemotaxis index, this method had a higher sensitivity for detecting early pancreatic cancer compared to existing diagnostic markers. The clinical application C. elegans for the early diagnosis of cancer can certainly be expected in the near future.

Impact of One-Carbon Metabolism-Driving Epitranscriptome as a Therapeutic Target for Gastrointestinal Cancer.

One-carbon (1C) metabolism plays a key role in biological functions linked to the folate cycle. These include nucleotide synthesis; the methylation of DNA, RNA, and proteins in the methionine cycle; and transsulfuration to maintain the redox condition of cancer stem cells in the tumor microenvironment. Recent studies have indicated that small therapeutic compounds affect the mitochondrial folate cycle, epitranscriptome (RNA methylation), and reactive oxygen species reactions in cancer cells. The epitranscriptome controls cellular biochemical reactions, but is also a platform for cell-to-cell interaction and cell transformation. We present an update of recent advances in the study of 1C metabolism related to cancer and demonstrate the areas where further research is needed. We also discuss approaches to therapeutic drug discovery using animal models and propose further steps toward developing precision cancer medicine.

Caenorhabditis elegans as a Diagnostic Aid for Pancreatic Cancer.

OBJECTIVES: Early detection of pancreatic cancer is notoriously difficult. A novel cancer diagnostic method using the ability of nematodes to detect odor of urine samples has been developed (N-NOSE). This method has a high sensitivity and specificity for various cancers; however, it has not yet been verified in pancreatic cancer. We examined the usefulness of this method to aid early diagnosis of pancreatic cancer in a cancer center. METHODS: We collected urine samples and clinical data from patients hospitalized in our division, between July 2017 and February 2019. We excluded patients with a known current or past history of other cancers. We investigated the relationship between the results of N-NOSE and the presence of pancreatic cancer. RESULTS: There were 95 noncancer cases and 104 pancreatic cancer cases. The sensitivity and specificity of N-NOSE for pancreatic cancer were 84.6% (88/104) and 60% (57/95), respectively. N-NOSE was able to detect stages 0 to I pancreatic cancer and had a higher correlation with early-stage pancreatic cancer than advanced stage. CONCLUSIONS: N-NOSE has sufficient sensitivity and specificity for use in clinical practice, and it holds great potential as a diagnostic aid for pancreatic cancer, especially for early-stage pancreatic cancer.

Accuracy evaluation of the C. elegans cancer test (N-NOSE) using a new combined method.

Early cancer detection is critical for effective treatment. N-NOSE (Nematode-NOSE) is a simple, inexpensive, and highly sensitive cancer screening method based on the chemotaxis of the nematode Caenorhabditis elegans, which shows evasive action from the urine of healthy individuals while being attracted to the urine of cancer patients. Initially, N-NOSE relied on chemotaxis indexes obtained with 10-fold dilutions of urine samples. However, cancer tissue size and concentrations of cancer odors differ among cancer patients. In this study, we examined the accuracy improvement of N-NOSE method by using two types of dilutions, 10-fold and 100-fold. We have conducted N-NOSE tests with urine samples from 32 cancer patients (esophageal, gastric, colorectal, gallbladder, cholangiocarcinoma, breast, malignant lymphoma, and acute myeloid leukemia) along with 143 healthy subjects. Our data showed a significant difference in the N-NOSE at 10-fold dilution between the two groups (p < 0.0001), with an area under the ROC curve (AUC) of 0.9188 based on receiver operating characteristic (ROC) analysis. N-NOSE index at 100-fold dilutions was also significantly different between the two groups (p < 0.0001), with an AUC of 0.9032 based on ROC analysis. In this clinical study, we further improve N-NOSE with a combined method of two dilutions (10-fold and 100-fold) of urine samples, which results in a markedly improvement in cancer detection sensitivity of 87.5%. N-NOSE sensitivity improvement was significantly high even for early-stage cancer detection, which is in stark contrast with the sensitivity of detection using blood tumor markers (CEA, CA19-9 and CA15-3). These results strongly suggest that the N-NOSE test by this new combined method strikes a good balance between sensitivity and specificity.

Immuno-Surgical Management of Pancreatic Cancer with Analysis of Cancer Exosomes.

Exosomes (EXs), a type of extracellular vesicles secreted from various cells and especially cancer cells, mesenchymal cells, macrophages and other cells in the tumor microenvironment (TME), are involved in biologically malignant behaviors of cancers. Recent studies have revealed that EXs contain microRNAs on their inside and express proteins and glycolipids on their outsides, every component of which plays a role in the transmission of genetic and/or epigenetic information in cell-to-cell communications. It is also known that miRNAs are involved in the signal transduction. Thus, EXs may be useful for monitoring the TME of tumor tissues and the invasion and metastasis, processes that are associated with patient survival. Because several solid tumors secrete immune checkpoint proteins, including programmed cell death-ligand 1, the EX-mediated mechanisms are suggested to be potent targets for monitoring patients. Therefore, a companion therapeutic approach against cancer metastasis to distant organs is proposed when surgical removal of the primary tumor is performed. However, EXs and immune checkpoint mechanisms in pancreatic cancer are not fully understood, we provide an update on the recent advances in this field and evidence that EXs will be useful for maximizing patient benefit in precision medicine.

State-of-the-Art Technology of Model Organisms for Current Human Medicine.

Since the 1980s, molecular biology has been used to investigate medical field mechanisms that still require the use of crude biological materials in order to achieve their necessary goals. Transcription factor-induced pluripotent stem cells are used in regenerative medicine to screen drugs and to support lost tissues. However, these cells insufficiently reconstruct whole organs and require various intact cells, such as damaged livers and diabetic pancreases. For efficient gene transfer in medical use, virally mediated gene transfers are used, although immunogenic issues are investigated. To obtain efficient detective and diagnostic power in intractable diseases, biological tools such as roundworms and zebrafish have been found to be useful for high-throughput screening (HST) and diagnosis. Taken together, this biological approach will help to fill the gaps between medical needs and novel innovations in the field of medicine.

COVID-19 Drug Discovery Using Intensive Approaches.

Since the infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China during December 2019, the coronavirus disease 2019 (COVID-19) has spread on a global scale, causing the World Health Organization (WHO) to issue a warning. While novel vaccines and drugs that target SARS-CoV-2 are under development, this review provides information on therapeutics which are under clinical trials or are proposed to antagonize SARS-CoV-2. Based on the information gained from the responses to other RNA coronaviruses, including the strains that cause severe acute respiratory syndrome (SARS)-coronaviruses and Middle East respiratory syndrome (MERS), drug repurposing might be a viable strategy. Since several antiviral therapies can inhibit viral replication cycles or relieve symptoms, mechanisms unique to RNA viruses will be important for the clinical development of antivirals against SARS-CoV-2. Given that several currently marketed drugs may be efficient therapeutic agents for severe COVID-19 cases, they may be beneficial for future viral pandemics and other infections caused by RNA viruses when standard treatments are unavailable.

Efficiency of Gastrointestinal Cancer Detection by Nematode-NOSE (N-NOSE).

BACKGROUND/AIM: Early detection of gastrointestinal cancer may reduce mortality. Recently, Caenorhabditis elegans has been reported to be capable of differentiating patients with cancers from healthy persons by the smell of urine. This novel technique using C. elegans olfaction has been named as Nematode-NOSE (N-NOSE). MATERIALS AND METHODS: We collected 180 urine samples from patients with gastrointestinal cancer and 76 samples from healthy subjects. N-NOSE test was performed using these samples and N-NOSE index was obtained. Quantification of the olfactory behavior of C. elegans was performed as established in past studies. By receiver operating characteristic (ROC) analysis, we examined the diagnostic capability of N-NOSE. RESULTS: ROC analysis revealed that N-NOSE showed an area under the curve value of more than 0.80, even in early-stage cancers. CONCLUSION: C. elegans olfaction enabled the detection of gastrointestinal cancers from urine with high sensitivity, which can provide the basis for the development of N-NOSE as a gastrointestinal cancer screening test.

Application of C. elegans cancer screening test for the detection of pancreatic tumor in genetically engineered mice.

Pancreatic ductal adenocarcinoma (PDAC) exhibits a very early onset of metastasis. Thus, early detection and treatment are pivotal to successful eradication of pancreatic cancers. Economical and non-invasive cancer screening systems is indispensable for this purpose. Previously our group developed a novel method to detect various kinds of human cancer using nematode Caenorhabditis elegans (C. elegans) that respond to cancer odor in urine; however, whether this method is useful for non-human species remains to be understood. In this study, we examined its effectiveness in the detection of murine pancreatic tumor spontaneously generated in genetically-engineered mice. We generated pancreas-specific Kras G12D and/or c-Met deletion mutant mice and measured the probability of spontaneous tumor generation in these mice. The chemotactic indexes of C. elegans to the urine samples of these mutant mice were measured. As previously described, oncogenic KrasG12D was necessary to induce pancreatic intraepithelial neoplasia in this mouse model, while c-Met mutation did not show further effect. The chemotactic analysis indicated that C. elegans avoids urine of healthy recipient mice, while they tended to be attracted to urine of mice with KrasG12D . Our study demonstrated that C. elegans can recognize the odor of pancreatic cancer in urine of KrasG12D model mouse, suggesting the similarity of cancer odor between species. Our result facilitates further studies on mechanism of cancer detection by C. elegans.

[Development of an Early Cancer Detection Method Using the Olfaction of the Nematode Caenorhabditis elegans].

Early detection and treatment are important for the successful eradication of various cancers; therefore, the development of economical, noninvasive novel cancer screening systems is critical. Previous reports using canine scent detection have demonstrated the existence of cancer-specific odors. However, it is difficult to introduce canine scent recognition into clinical practice because of the need to maintain accuracy. In this study, we developed a Nematode-Nose (N-NOSE) test using Caenorhabditis elegans to provide a novel, highly accurate cancer detection system that is economical, painless, rapid, and convenient. We demonstrated that wild-type C. elegans displayed attractive chemotaxis toward human cancer cell secretions, cancer tissues, and urine from cancer patients but avoided control urine. In parallel, C. elegans olfactory neurons showed a significantly stronger response to urine from cancer patients than to control urine. In contrast, G protein α mutants and animals with ablated olfactory neurons were not attracted to urine from cancer patients, suggesting that they sense odors in urine. We tested 242 samples to measure the performance of the N-NOSE test and found that the sensitivity was 95.8%, which is markedly higher than that of other existing tumor markers. Furthermore, the specificity was 95.0%. Importantly, this test could detect various cancer types tested at the early stage (stage 0 or 1). C. elegans scent-based analyses therefore might provide a new strategy for the detection and study of disease-associated scents.

Behavioural Response Alteration in Caenorhabditis elegans to Urine After Surgical Removal of Cancer: Nematode-NOSE (N-NOSE) for Postoperative Evaluation.

The technique used for cancer monitoring is essential for effective cancer therapy. Currently, several methods such as diagnostic imaging and biochemical markers have been used for cancer monitoring, but these are invasive and show low sensitivity. A previous study reported that Caenorhabditis elegans sensitively discriminated patients with cancer from healthy subjects, based on the smell of a urine sample. However, whether C. elegans olfaction can detect the removal of cancerous tumours remains unknown. This study was conducted to examine C. elegans olfactory behaviour to urine samples collected from 78 patients before and after surgery. The diagnostic ability of the technique termed Nematode-NOSE (N-NOSE) was evaluated by receiver operating characteristic (ROC) analysis. The ROC curve of N-NOSE was higher than those of classic tumour markers. Furthermore, we examined the change in C. elegans olfactory behaviour following exposure to preoperative and postoperative samples. The results suggest that a reduction in attraction indicates the removal of the cancerous tumour. This study may lead to the development of a noninvasive and highly sensitive tool for evaluating postoperative cancer patients.

[Early Diagnosis of Cancer Using Nematoda C. elegans].

Cancer is the leading cause of death in Japan, and one in two people experience cancer in their lifetime. Early diagnosis of cancer is the most important for increasing survival rate of cancer, which is also expected to contribute to decrease budget impact of cancer, but participation rate of cancer screening is still low in Japan. Currently, people need to take multiple examinations to detect different types of cancer, which increases the cost, time and pain burdens for the examinees. Therefore, it is desirable to develop cheaper, non-invasive, as well as sensitive cancer screening methods that can detect multiple types of cancer at the same time. Most of the existing cancer screening tests including imaging diagnosis depend on artificial devices, which usually require high cost to achieve high sensitivity. We have developed a new technique, N-NOSE, which takes advantage of the good olfaction of nematode C. elegans to detect cancer smell in urine samples. N-NOSEexhibited 95.8% sensitivity and 95.0% specificity on 242 urine samples of 10 cancers types tested including those of early stages. C. elegans is easy to be maintained in a laboratory with low cost. In addition, as C. elegans is a hermaphroditic organism with homogeneous genetic background, they show stable and reproducible behavioral results. Therefore, N-NOSEis expected to offer a reasonable and non-invasive cancer screening method which is suitable for regular health checkup.

Establishment of Time- and Cell-Specific RNAi in Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans, in which loss-of-function mutants and RNA interference (RNAi) models are available, is a model organism useful for analyzing effects of genes on various life phenomena. In particular, RNAi is a powerful tool that enables time- or cell-specific knockdown via heat shock-inducible RNAi or cell-specific RNAi. However, the conventional RNAi methods are insufficient for investigating pleiotropic genes with various sites of action and life stage-dependent functions. To investigate the temporal- and cell-specific profiles of multifunctional genes, we established a new RNAi method that enables simultaneous time- and cell-specific knockdown (T.C.RNAi) in C. elegans. In this method, one RNA strand is expressed by a cell-specific promoter and the other by a heat shock promoter, resulting in only expression of double-stranded RNA in the target cell when heat shock is induced. We confirmed the effect of T.C.RNAi by the knockdown of GFP and the odr-3 gene which encodes Gα and is essential for olfaction. Further, this technique revealed that the control of glutamate receptors GLR-1 localization in RMD motor neurons requires Ras at the adult stage to regulate locomotion behavior.